Beta amyloid 40 and beta amyloid 42 specific recognition polypeptides and uses thereof

Abstract

This invention discloses a group of Aβ40 and Aβ42-specific recognition peptides, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The invention also discloses conjugates of the Aβ40 and Aβ42-specific recognition peptides with carrier proteins, their applications, and the preparation of immunochromatographic detection reagents or kits for Alzheimer's disease. Compared to existing technologies, using magnetic microparticles coated or blocked with the peptides shown in SEQ ID NO.1 and 2 of this invention, under stable reagent components and processes, can effectively improve the sensitivity of immunochromatographic detection reagents; furthermore, the peptides shown in SEQ ID NO.1 and 2 do not or almost do not cross-react with other types of β-amyloid proteins, thus simultaneously ensuring both sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic detection technology, specifically, it relates to a group of peptides that specifically recognize β-amyloid 40 and β-amyloid 42 and their applications. Background Technology

[0002] Alzheimer's disease is the most common neurodegenerative disease, and its incidence rate is increasing year by year, making it the leading cause of death among the elderly. In recent years, the demand for early diagnostic reagents for Alzheimer's disease has been increasing both internationally and domestically. Current main diagnostic methods include cerebrospinal fluid testing and imaging diagnosis, but these methods are either invasive or expensive. In contrast, chemiluminescence and immunochromatography are the most widely used diagnostic methods, offering advantages such as high throughput, low cost, and non-invasiveness. The main bottleneck in applying chemiluminescence and immunochromatography to the early blood diagnosis of Alzheimer's disease is improving the sensitivity and precision of the reagents.

[0003] Currently, the sensitivity requirements for detecting the concentrations of β-amyloid 40 (Aβ40) and β-amyloid 42 (Aβ42) in blood samples using chemiluminescence are approaching the methodological limits. Except for Aβ, the concentrations of other Alzheimer's disease biomarkers in blood are even lower, placing increasingly higher demands on reagents, antibodies, raw materials, and processes. Therefore, breakthroughs in these areas are crucial for improving chemiluminescence sensitivity and providing effective, inexpensive, and rapid in vitro diagnostic reagents for Alzheimer's disease. Existing or under-development Alzheimer's disease blood immunodiagnostic reagents mainly rely on high-affinity antibodies as biological raw materials, using a double-antibody sandwich method to quantitatively analyze the concentration of biomarkers in the blood. However, due to the large molecular weight of antibodies, the binding efficiency is limited during solid-phase coating. Furthermore, antibody orientation during coating can also affect the reagent's sensitivity to some extent.

[0004] In summary, it is necessary to develop biomaterials for immunodiagnostics, including but not limited to antibodies, through different approaches, so as to further promote the clinical diagnosis of Alzheimer's disease and other neurodegenerative diseases. Summary of the Invention

[0005] The purpose of this invention is to improve existing immunochromatographic detection methods for the early clinical diagnosis of Alzheimer's disease, so as to enhance the detection sensitivity of Alzheimer's disease blood markers Aβ40 and Aβ42.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a set of Aβ40 and Aβ42 specific recognition peptides, the sequence of the Aβ40 specific recognition peptide being shown in SEQ ID NO.1 and the sequence of the Aβ42 specific recognition peptide being shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention provides a set of conjugates of Aβ40 and Aβ42 specific recognition peptides and carrier proteins, wherein the sequence of the Aβ40 specific recognition peptide is shown in SEQ ID NO.1 and the sequence of the Aβ42 specific recognition peptide is shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention provides the application of the aforementioned group of Aβ40 and Aβ42 specific recognition peptides for the preparation of immunochromatographic detection reagents or kits for Alzheimer's disease, wherein the peptide shown in SEQ ID NO.1 is used for the detection of Aβ40, and the peptide shown in SEQ ID NO.2 is used for the detection of Aβ42.

[0009] According to the present invention, the application involves using conjugates of peptides and carrier proteins shown in SEQ ID NO. 1 and 2 coated with magnetic microparticle reagents, which are then mixed with magnetic microparticle reagents coated with Aβ40 and Aβ42 antibodies, respectively, and then used in immunochromatographic detection reagents or kits.

[0010] According to a preferred embodiment of the present invention, the magnetic microparticle reagent coated with the conjugate of the polypeptide and carrier protein shown in SEQ ID NO.1 and 2 is mixed with the magnetic microparticle reagent coated with Aβ40 or Aβ42 antibody in a volume ratio of 1:4.

[0011] According to the present invention, the magnetic microparticles coated with the conjugates of the polypeptides and carrier proteins shown in SEQ ID NO.1 and 2 and the magnetic microparticles coated with Aβ40 and Aβ42 antibodies are both blocked with conventional blocking buffer.

[0012] According to a preferred embodiment, the magnetic microparticles coated with the conjugates of the peptides and carrier proteins shown in SEQ ID NO. 1 and 2 are blocked with conventional blocking buffer; the blocking buffer for blocking the magnetic microparticles coated with Aβ40 antibody is a conventional blocking buffer supplemented with 0.05±0.01wt% of the conjugate of the peptides and carrier proteins shown in SEQ ID NO. 1; the blocking buffer for blocking the magnetic microparticles coated with Aβ42 antibody is a conventional blocking buffer supplemented with 0.05±0.01wt% of the conjugate of the peptides and carrier proteins shown in SEQ ID NO. 2.

[0013] According to another preferred embodiment, the application is: adding the conjugate of the polypeptide and carrier protein shown in SEQ ID NO.1 to a blocking buffer to block magnetic microparticles coated with Aβ40 antibody; and adding the conjugate of the polypeptide and carrier protein shown in SEQ ID NO.2 to a blocking buffer to block magnetic microparticles coated with Aβ42 antibody.

[0014] Furthermore, the amount of the conjugate of the polypeptide and carrier protein shown in SEQ ID NO.1 or 2 added is 0.05 ± 0.01 wt%.

[0015] In a fourth aspect, the present invention provides a magnetic microparticle blocking buffer, wherein, by weight percentage, 0.05 ± 0.01% of a conjugate of the polypeptide and carrier protein shown in SEQ ID NO. 1 or 2 is added to the blocking buffer.

[0016] In a fifth aspect, the present invention provides an immunochromatographic detection reagent or kit for Alzheimer's disease, the reagent or kit comprising magnetic microparticle reagents coated with conjugates of peptides and carrier proteins shown in SEQ ID NO. 1 and 2, wherein the peptide shown in SEQ ID NO. 1 is used for the detection of Aβ40 and the peptide shown in SEQ ID NO. 2 is used for the detection of Aβ42.

[0017] A sixth aspect of the present invention provides an immunochromatographic detection reagent or kit for Alzheimer's disease, the reagent or kit comprising a magnetic microparticle reagent coated with Aβ40 or Aβ42 antibody and blocked using the magnetic microparticle blocking buffer described above.

[0018] The present invention has the following beneficial effects:

[0019] 1. Compared with existing technologies, the use of the peptide-coated or blocked magnetic microparticles shown in SEQ ID NO.1 and 2 of the present invention can effectively improve the sensitivity of immunochromatographic detection reagents when the reagent components and processes are stable.

[0020] 2. The polypeptides shown in SEQ ID NO.1 and 2 of the present invention do not or hardly cross-react with other types of β-amyloid proteins, thus ensuring both sensitivity and specificity. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] Unless otherwise specified, all percentage concentrations mentioned in the following examples are mass percentage concentrations.

[0023] In the following examples, peptide 1 (sequence: SGYHHFAEDVGAIIGLMGGVV, used for the detection of Aβ40) shown in SEQ ID NO.1 and peptide 2 (sequence: SGYHHFAEDVGAIMGVVIA, used for the detection of Aβ42) shown in SEQ ID NO.2 are both synthetic sequences. These sequences are mainly the aggregation core sequences of Alzheimer's disease biomarkers β-amyloid protein 40 (Aβ40) and 42 (Aβ42) when they form a deposition state.

[0024] During synthesis, an NHS active group was added to the N-terminus and dissolved in DMSO at a concentration of 10 mg / mL. After dissolution, the peptide was diluted to 1 mg / mL with 20 mM HEPES buffer (pH 8.0), and then conjugated with bovine serum albumin (BSA), casein, and human gamma globulin (hIgG) at a molar ratio of 20:1 using 20 mM HEPES (pH 8.0) as the conjugation buffer. The reaction was carried out overnight at 4°C. After conjugation, unreacted peptides were separated using a desalting column, and the concentration of the peptide-BSA conjugate was determined using the BCA method.

[0025] For direct coating, add 20 μg of peptide per mg of magnetic microparticles; for blocking, add an additional 0.05 ± 0.01% peptide-BSA polymer to the blocking buffer.

[0026] During peptide reactivity verification, a series of samples diluted at different concentrations and 10 random normal human serum samples were tested. According to the corresponding test content, except for the difference in the magnetic microparticle addition ratio or magnetic microparticle blocking buffer, the other steps were kept consistent for each group.

[0027] All samples were compared with a blank buffer solution to eliminate systematic errors and facilitate sensitivity analysis.

[0028] Unless otherwise specified, HEPES, bovine serum albumin (BSA), casein-Na, Tween-type emulsifier, and biological preservatives in the following examples are all commercially available. The blocking and preservation solution was prepared using conventional mixed solution preparation methods. The fully automated chemiluminescence immunoassay analyzer Shine i1910 used for immunofluorescence detection is a commercially available product.

[0029] Example 1: Preparation of β-amyloid protein detection reagent

[0030] 1.1 Preparation of R1 reagent

[0031] In this embodiment, reagent R1 includes: magnetic microparticles coated with Aβ40 specific antibody (magnetic microparticle 1), magnetic microparticles coated with Aβ42 specific antibody (magnetic microparticle 2), magnetic microparticles coated with polypeptide 1 (magnetic microparticle 3), and magnetic microparticles coated with polypeptide 2 (magnetic microparticle 4).

[0032] The preparation method of reagent R1 is as follows:

[0033] Dialyze the Aβ40 antibody overnight at 4°C using 0.05 mol / L sodium bicarbonate solution. Change the buffer at least three times during dialysis. After dialysis, remove the Aβ40 antibody, determine its concentration, and bring the volume to 1 mg / mL with 0.05 M sodium bicarbonate buffer. Store at 4°C. Wash the magnetic microparticles to be prepared at least three times with 0.10 mol / L MES buffer. Add the required amount of magnetic microparticles and antibody at a ratio of 10.00–40.00 μg of Aβ40 antibody per 1.00 mg of magnetic microparticles. Rotate the mixture at room temperature for 3–5 hours to obtain magnetic microparticles coated with Aβ40 antibody (magnetic microparticle 1).

[0034] Following the same method described above, the Aβ40 antibody was replaced with the Aβ42 antibody, the polypeptide 1-BSA conjugate, and the polypeptide 2-BSA conjugate, respectively, to obtain magnetic microparticles coated with the Aβ42 antibody (magnetic microparticle 2), magnetic microparticles coated with the polypeptide 1-BSA conjugate (magnetic microparticle 3), and magnetic microparticles coated with the polypeptide 2-BSA conjugate (magnetic microparticle 4).

[0035] The magnetic microparticles 1–4 were blocked using two different blocking buffer solutions, as detailed below:

[0036] The formulations for the two blocking buffers are as follows:

[0037] Blocking buffer 1 uses a standard blocking buffer formulation prepared with deionized water: Tris 50mM, NaCl 50mM, BSA 0.5%, Proclin-300 0.03%, EDTA 1%, Tween-20 0.1%, and Casein 0.5%.

[0038] The formulation of blocking buffer 2 is as follows: Based on blocking buffer 1, add 0.05±0.01% of peptide 1-BSA conjugate or peptide 2-BSA conjugate.

[0039] The two closure schemes are as follows:

[0040] Option 1: Block magnetic microparticles 1 coated with Aβ40, magnetic microparticles 2 coated with Aβ42 antibody, magnetic microparticles 3 coated with peptide 1-BSA conjugate, and magnetic microparticles 4 coated with peptide 2-BSA conjugate using the above-mentioned blocking buffer 1, and block at 4°C for 14-18 hours.

[0041] Option 2: Block magnetic microparticles 1 coated with Aβ40 and magnetic microparticles 2 coated with Aβ42 antibody using the above-mentioned blocking buffer 2, and block at 4°C for 14-18 hours.

[0042] After sealing, the magnetic microparticles were separated from the liquid fraction using a magnetic separation plate. The magnetic microparticles were washed at least twice with MES buffer. The solution was then transferred to reagent buffer and brought to a final volume of 10 mg magnetic microparticles / mL, designated as the corresponding concentrated solutions of sealed magnetic microparticles 1–4, and stored at 4°C for later use. Thorough mixing is required before use.

[0043] The reagent buffer solution is formulated (prepared with deionized water): Tris 0.10 mol / L, BSA 1.00%, NaCl 0.90%, Casein-Na 0.50%, Proclin-300 0.10%, Triton X-405 0.01%, Bovine serum gamma globulin (BGG) 1.00%, Tween-20 0.50%, EDTA 0.20%, antifoaming agent 0.10%, gentamicin sulfate 0.01%, and Sucrose 15.00%.

[0044] 1.2 Preparation of R2 reagent

[0045] In this embodiment, reagent R2 is an Aβ40 / 42 antibody labeled with acridinium ester, and its preparation process is as follows:

[0046] Dialyze Aβ40 / 42 antibody overnight at 4°C using 0.05 mol / L sodium bicarbonate solution. Change the buffer at least three times during dialysis. After dialysis, remove the antibody, determine its concentration, and add 0.05 M sodium bicarbonate buffer as needed to control the antibody at a suitable final concentration. Then store at 4°C.

[0047] Add Aβ40 / 42 antibody and acridine ester to a brown reaction flask at a reaction concentration of 0.1 mg acridine ester per 1 mg of antibody, under light-protected conditions. Incubate at room temperature with shaking for 1 hour in the dark.

[0048] Prepare a 10.00% glycine aqueous solution. In a light-protected environment, add the corresponding volume of glycine solution to the reaction flask at a reaction concentration of 1 mL of glycine solution per 1 mg of Aβ40 / 42 antibody. Incubate at room temperature with shaking for 1–2 hours in the dark.

[0049] After the reaction is complete, take a 3.5 kDa dialysis bag of appropriate length that is soaked in ethanol solution, add all the liquid from all the reaction flasks into the dialysis bag, and dialyze using 0.05 mol / L sodium bicarbonate buffer at 4°C in the dark. Change the solution at least three times during the dialysis process, with an interval of no less than 4 hours between each solution change.

[0050] After dialysis, the concentrated solution of acridine ester Aβ40 / 42 antibody was added to the solution and the concentration was determined using the BCA method.

[0051] Because acridine esters are photosensitive, they must be stored at -20°C in the dark. Remove from the container at least 30 minutes before use, store in the dark, and allow to return to room temperature. Avoid generating air / liquid bubbles when shaking.

[0052] Example 2: Reagent Performance Evaluation

[0053] 2.1 Mixing ratio of magnetic microparticles and polypeptide-BSA coupled magnetic microparticles

[0054] The purpose of this embodiment is to screen the optimal mixing ratio of magnetic microparticles and peptide-BSA coupled magnetic microparticles.

[0055] Experimental Protocol: In this embodiment, the non-specific immunoreaction of the peptides and BSA conjugates (peptide 1-BSA conjugate and peptide 2-BSA conjugate) of SEQ ID NO. 1 and SEQ ID NO. 2 was verified using R1 reagents (magnetic microparticles 1 coated with Aβ40, magnetic microparticles 2 coated with Aβ42 antibody, magnetic microparticles 3 coated with peptide 1-BSA conjugate, and magnetic microparticles 4 coated with peptide 2-BSA conjugate) prepared in Example 1 and blocked with blocking buffer 1 was verified. Magnetic microparticles 1 and 2 were diluted to preset concentrations using fetal bovine serum, and magnetic microparticles 1 or 2 were mixed with magnetic microparticles 3 or 4 according to the volume ratios shown in Table 1, and prepared using the reagent buffer described in 1.2 to ensure the same final concentration of magnetic microparticles.

[0056] Table 1: Mixing ratio of magnetic microparticles: peptide-BSA coupled magnetic microparticles

[0057] Group number Magnetic particle 1: Magnetic particle 3 Magnetic particle 2: Magnetic particle 4 Comparison Magnetic particle 1 (non-magnetic particle 3) Magnetic particles 2 (non-magnetic particles 4) #1 1:1 1:1 #2 2:1 2:1 #3 4:1 4:1 #4 8:1 8:1 #5 1:2 1:2 #6 1:4 1:4 #7 1:8 1:8

[0058] Testing plan:

[0059] The relative luminescence intensity (RLU) of serially diluted samples (#1-7) and the control group was measured three times per concentration point. The mean (M) of the three RLU measurements was calculated, and the ratio of the mean (M) to the mean (M) of the relative luminescence intensity (RLU) of the blank value (i.e., P / N) was calculated. At the same concentration, a higher relative luminescence intensity (RLU) and a larger P / N indicate better reagent performance under those conditions.

[0060] The detection was performed using a fully automated chemiluminescence analyzer, employing the following general detection modes:

[0061] Step 1: Add 50 μL of sample to the reaction tube provided with the instrument;

[0062] Step 2: Add 50 μL of R1 reagent to the reaction tube provided with the instrument;

[0063] Step 3: Add 50 μL of R2 reagent to the reaction tube provided with the instrument;

[0064] Step 4: After mixing, incubate at 37.00±1.00℃ for 15 minutes;

[0065] Step 5: The instrument automatically performs magnetic separation and discards the supernatant;

[0066] Step 6: The instrument automatically adds the matching pre-activation solution and activation solution according to the program, and then mixes and incubates them.

[0067] Step 7: Detect the luminescence intensity.

[0068] The test results are shown in Tables 2-1 and 2-2.

[0069] Table 2-1: Optimal ratio screening data for Aβ40 immunoassay

[0070]

[0071] Table 2-2: Optimal ratio screening data for Aβ42 immunoassay

[0072]

[0073]

[0074] As shown in Tables 2-1 and 2-2, in the Aβ40 / 42 immunoassay, when reagent R1 was mixed with magnetic microparticles 1:3 and 2:4 in a 4:1 ratio, the detection signal values ​​and sensitivity of Aβ peptides at all concentrations were significantly improved compared to the control, even without a significant change in background signal value. Changing the ratio negatively impacted the relative luminescence intensity (RLU) measurement compared to the control group, but the discrimination remained somewhat improved. Furthermore, the results indicate that for the Aβ40 / 42 immunoassay, especially the Aβ42 immunoassay, adjusting the ratio of magnetic microparticles 2:4 to 4 in a 4:1 ratio significantly improved the P / N ratio at the lowest concentration point (1 pg / mL), helping the reagent to stably distinguish samples at that concentration.

[0075] 2.2 Evaluation of the impact of different blocking buffers on reagent performance

[0076] The purpose of this embodiment is to evaluate the effect of different blocking buffers (blocking buffer 1 and blocking buffer 2) on reagent performance while keeping other conditions consistent.

[0077] Experimental protocol: Same as described in 2.1. The difference is that this experiment only examines the effects of different blocking buffers (blocking buffer 1 and blocking buffer 2) on magnetic particles 1 and 2, and does not involve magnetic particles 3 and 4.

[0078] The detection mode is the same as 2.1.

[0079] The test results are shown in Tables 3-1 and 3-2.

[0080] Table 3-1: The impact of changes in the blocking buffer for Aβ40 immunoassay on reagent performance

[0081]

[0082]

[0083] Table 3-2: The impact of changes in the blocking buffer for Aβ42 immunoassay on reagent performance

[0084]

[0085] As shown in Tables 3-1 and 3-2, compared with magnetic microparticles blocked using conventional blocking buffer 1, blocking with blocking buffer 2 resulted in an approximately 30% increase in both the detection signal value and sensitivity of Aβ peptides at all concentrations. Furthermore, compared with blocking buffer 1, blocking with blocking buffer 2 significantly improved the discrimination of the magnetic microparticle reagent at the minimum concentration point, indicating that blocking with blocking buffer 2 significantly improves reagent performance.

[0086] 2.3 Sample Detection Performance Evaluation

[0087] The purpose of this embodiment is to evaluate the improvement in sample performance under optimal conditions compared to the control group.

[0088] Experimental Protocol: Based on the experimental conclusions in 2.1 and 2.2, magnetic microparticles 1 and 2 coated with Aβ40 and Aβ42, prepared using blocking buffer 2, were mixed with magnetic microparticles 3 and 4 coated with peptide 1-BSA coupling agent and peptide 2-BSA coupling agent, respectively, at a ratio of 4:1. The mixture was prepared using reagent buffer 1.2, and 10 random samples were tested. Fetal bovine serum was used as a blank control. The ratio (S / N) between the relative luminescence intensity (RLU) measured in the sample and the relative luminescence intensity (RLU) measured in the blank control was calculated. A stronger relative luminescence intensity (RLU) and a higher S / N for the same sample indicated better reagent performance. The evaluation groups and detailed conditions are shown in Table 4.

[0089] Table 4: Reagent Performance Evaluation Group Settings

[0090]

[0091] The detection mode is the same as 2.1.

[0092] The test results are shown in Tables 5-1 and 5-2.

[0093] Table 5-1: Aβ40 Immunoassay Sample Measurement Data

[0094]

[0095]

[0096] Table 5-2: Aβ42 Immunoassay Sample Data

[0097]

[0098] As shown in Tables 5-1 and 5-2, in the Aβ40 / 42 immunoassay reagent, when the blocking buffer is adjusted according to the preparation process of this invention and magnetic microparticles 3 or 4 coated or blocked by peptide 1-BSA or peptide 2-BSA conjugate are added, the relative luminescence intensity (RLU) of the blank control is close to or slightly lower than that of the control group. However, the relative luminescence intensity (RLU) of the random sample is close to or higher than that of the control. This indicates that adding peptide 1-BSA or peptide 2-BSA conjugate to coat the magnetic microparticles and replacing half of the blocking buffer with the magnetic microparticles coated with Aβ40 / 42 can improve the signal value and sensitivity of the reagent.

[0099] 2.4 Precision Evaluation

[0100] The purpose of this embodiment is to evaluate the difference in precision between the implementation group and the control group under optimal conditions.

[0101] Experimental protocol: Test the sample dilution buffer or fetal bovine serum. Repeat the test of the same bottle of buffer or fetal bovine serum 20 times for each reagent group. Calculate the mean (M) and standard deviation (SD) of relative luminescence intensity (RLU) and calculate the coefficient of variation (CV%).

[0102] When all other conditions except the blocking buffer remain unchanged, the smaller the average (M) of the relative luminescence intensity (RLU) measured by the reagent in the sample dilution buffer or fetal bovine serum, and the smaller the coefficient of variation (CV%), the better the reagent performance. The evaluation group settings are the same as those in Table 4 in 2.3.

[0103] The detection mode is the same as 2.1.

[0104] The test results are shown in Tables 6-1 and 6-2.

[0105] Table 6-1: Repeatability Evaluation Experimental Data of Aβ40 Immunoassay Reagent

[0106]

[0107]

[0108] Table 6-2: Repeatability Evaluation Experimental Data of Aβ42 Immunoassay Reagent

[0109]

[0110] As shown in Tables 6-1 and 6-2, in the Aβ40 / 42 immunoassay, the groups to be evaluated showed the same reproducibility. Meanwhile, the magnetic microparticles 1 or 2 blocked with blocking buffer 2 and magnetic microparticles 3 or 4 with peptide 1-BSA or peptide 2-conjugate added showed the best reagent reproducibility.

[0111] 2.5 Sensitivity Evaluation

[0112] The purpose of this embodiment is to evaluate the difference in sensitivity between the implementation group and the control group under optimal conditions.

[0113] Experimental protocol: Dilute the recombinantly expressed antigen with fetal bovine serum and measure the serially diluted samples, with each measurement performed twice. Calculate the ratio (P / N) between the average relative luminescence intensity (RLU) measured at each point and the average relative luminescence intensity (RLU) measured in the buffer solution.

[0114] When all other conditions except the blocking solution remain constant, at the same concentration point, a stronger relative luminescence intensity (RLU) and a higher P / N ratio indicate better reagent performance. The evaluation groups are set as shown in Table 4 in section 2.3.

[0115] The detection mode is the same as 2.1.

[0116] The test results are shown in Tables 7-1 and 7-2.

[0117] Table 7-1: Experimental data on sensitivity evaluation of Aβ40 immunoassay reagent

[0118]

[0119] Table 7-2: Experimental data on sensitivity evaluation of Aβ42 immunoassay reagent

[0120]

[0121] As shown in Tables 7-1 and 7-2, the Aβ40 / 42 immunoassay reagent maintained good signals for samples at different gradients. While the overall signal level was slightly lower than the control, the overall discrimination was good. Group #3, which used blocking buffer 2 to block magnetic microparticles 1 or 2 and added peptide 1-BSA or peptide 2-conjugate magnetic microparticles 3 or 4, exhibited good sensitivity and discrimination across all concentration ranges due to its high overall signal level, even with a background similar to the other groups.

[0122] 2.6 Evaluation of Cross-Reactivity

[0123] The purpose of this embodiment is to evaluate whether there is cross-reactivity between the Aβ40 / 42 project implementation group and the control group and the Aβ42 / 40 protein under optimal conditions.

[0124] Experimental protocol: Dilute the recombinantly expressed antigen to 10 pg / mL using calf serum, and add concentrations of 0, 10, 30, 100, 300, and 1000 pg / mL of the present invention for detecting Aβ42 / 40 polypeptide 1 / 2 to observe whether it affects the detection signal and discrimination during the assay.

[0125] The absolute value of the relative luminescence intensity (RLU) measured when the peptide 1 / 2 addition concentration is ≤100 pg / mL should not deviate ≥5% from the relative luminescence intensity (RLU) measured when the peptide 1 / 2 addition concentration is 0 pg / mL (i.e., no peptide added). The evaluation group settings are the same as those in Table 4 in 2.3.

[0126] The detection mode is the same as 2.1.

[0127] The test results are shown in Tables 8-1 and 8-2.

[0128] Table 8-1: Experimental data on cross-reactivity evaluation of Aβ40 immunoassay reagents

[0129]

[0130] Table 8-2: Experimental data on cross-reactivity evaluation of Aβ42 immunoassay reagents

[0131]

[0132] As shown in Tables 8-1 and 8-2, when different concentrations of the Aβ40 / 40 peptide 1 / 2 of this invention were added to diluted samples with a corresponding concentration of 10 pg / mL, a significant change in the discrimination between the standard and the background (i.e., the absolute value of the relative luminescence intensity (RLU) deviation ≥ 5%) only appeared when the added amount reached 300 pg / mL. At an added amount of 300 pg / mL, the effect on the measurement results was 5–20%; at an added amount of 1000 pg / mL, the effect was 20–40%. Changes in discrimination at other concentrations should be understood as due to instrument fluctuations, and are not considered to have a significant impact on the reagent measurement results within this concentration range.

[0133] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A specific recognition polypeptide designed based on the core sequence of β-amyloid aggregation, characterized in that, The polypeptide is selected from: Aβ40-specific recognition polypeptide with a sequence as shown in SEQ ID NO.1; or The Aβ42-specific recognition polypeptide has the sequence shown in SEQ ID NO.

2.

2. The use of the specific recognition peptide according to claim 1 in the preparation of a chemiluminescent immunoassay reagent or kit for the auxiliary detection of Aβ40 or Aβ42; wherein, The Aβ40-specific recognition polypeptide, as shown in SEQ ID NO.1, is used to assist in the detection of Aβ40. The Aβ42-specific recognition polypeptide, as shown in SEQ ID NO.2, is used to assist in the detection of Aβ42.

3. A conjugate of a specific recognition peptide and a carrier protein designed based on the core sequence of β-amyloid aggregation, characterized in that, The coupling element is selected from: A conjugate of an Aβ40-specific recognition polypeptide and a carrier protein with the sequence shown in SEQ ID NO.1; or A conjugate of an Aβ42-specific recognition polypeptide and a carrier protein, as shown in SEQ ID NO.

2.

4. The use of the conjugate according to claim 3 in the preparation of a chemiluminescent immunoassay reagent or kit for the auxiliary detection of Aβ40 or Aβ42; wherein, The conjugate of the Aβ40-specific recognition polypeptide with the sequence shown in SEQ ID NO.1 and the carrier protein is used to assist in the detection of Aβ40; The conjugate of the Aβ42-specific recognition polypeptide with the sequence shown in SEQ ID NO.2 and the carrier protein is used to assist in the detection of Aβ42.

5. The application according to claim 4, characterized in that, The application includes: mixing magnetic microparticles coated with the polypeptide-carrier protein conjugate shown in SEQ ID NO.1 and magnetic microparticles coated with Aβ40 antibody at a volume ratio of 1:4 for the detection of Aβ40; or Magnetic microparticles coated with the polypeptide and carrier protein conjugate shown in SEQ ID NO.2 were mixed with magnetic microparticles coated with Aβ42 antibody at a volume ratio of 1:4 for the detection of Aβ42.

6. The application according to claim 5, characterized in that, The magnetic microparticles coated with Aβ40 antibody were blocked using a blocking buffer containing 0.05 ± 0.01 wt% of the conjugate. The conjugate is a conjugate of the polypeptide shown in SEQ ID NO.1 and a carrier protein.

7. The application according to claim 5, characterized in that, The magnetic microparticles coated with Aβ42 antibody were blocked using a blocking buffer containing 0.05 ± 0.01 wt% of the conjugate. The conjugate is a conjugate of the polypeptide shown in SEQ ID NO.2 and a carrier protein.

8. A magnetic microparticle blocking buffer solution, characterized in that, The blocking buffer contains 0.05 ± 0.01 wt% of the conjugate as described in claim 3 by weight percentage.

9. A chemiluminescent immunoassay reagent or kit for the auxiliary detection of Aβ40 or Aβ42, characterized in that, It comprises magnetic microparticles coated with the coupling as described in claim 3.

10. A chemiluminescent immunoassay reagent or kit for detecting Aβ40 or Aβ42, characterized in that, It includes magnetic microparticles blocked using the magnetic microparticle blocking buffer of claim 8 and coated with Aβ40 or Aβ42 antibody.

Images